β-lactam compounds and process for preparing the same

ABSTRACT

A β-lactam compound of the formula:                    
     wherein R 1  is lower alkyl or OH-substituted lower alkyl, R 2  is H or lower alkyl, X is O, S or NH, n is 1 to 3, R 3  is —C(R a )═NH (R a  is H, lower alkyl or substituted lower alkyl), or a salt thereof, or an ester thereof. These compounds show excellent antibacterial activity against Gram-positive bacteria, particularly against methicillin-resistant Staphylococci and methicillin-resistant and coagulase-negative Staphylococci.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP97/03078 which has an Internationalfiling date of Sep. 3, 1997 which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a novel β-lactam compound representedby the formula [1] as described below.

BACKGROUND ART

By the wide clinical application of the third-generation cephalosporins,Gram-positive bacteria have become to be frequently isolated.Particularly, methicillin-resistant Staphylococcus aureus (hereinafter,referred to as MRSA) has been more frequently isolated, and becomes aserious problem in clinical field, because infectious diseases caused byMRSA are difficult to be treated. For example, vancomycin, which hasbeen broadly used for infectious diseases caused by MRSA in these days,is difficult to be administered because of its side effects, andglycopeptide-resistant bacteria are supposed to increase in future byadministration thereof. Moreover, it has recently been reported theincrease in isolation of methicillin-resistant and coagulase-negativeStaphylococci (MRCNS). Under these circumstances, it has been desired todevelop a safe drug having potent anti-MRSA and anti-MRCNS activities.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a β-lactam antibiotichaving an excellent antibacterial activity against Gram-positivebacteria, especially against MRSA and MRCNS.

The present inventors have intensively studied, and have found that acompound of the following formula [1] shows a potent effect onGram-positive bacteria, and shows an excellent antibacterial activityespecially against MRSA and MRCNS, and have accomplished the presentinvention.

That is, the present invention relates to a compound of the formula [1]:

wherein R¹ is a lower alkyl group or a lower alkyl group beingsubstituted by a hydroxy group, R² is a hydrogen atom or a lower alkylgroup, X is O, S or NH, n is 1 to 3, and R³ is —C(R^(a))═NH, in whichR^(a) is a hydrogen atom, a lower alkyl group, or a substituted loweralkyl group, or a pharmaceutically acceptable salt thereof, or anon-toxic ester thereof.

Moreover, the present invention also relates to a process for preparinga compound of the formula [1]:

wherein R¹, R², R³ ₁, X and n are the same as defined above, or apharmaceutically acceptable salt thereof, or a non-toxic ester thereof,which comprises

reacting a compound of the formula [2]:

wherein R² is the same as defined above, R^(1a) is a lower alkyl group,a lower alkyl group being substituted by a hydroxy group, or a loweralkyl group being substituted by a hydroxy group protected by aprotecting group, R⁴ is a protecting group for carboxyl group, and L isan active ester of hydroxy group, with a compound of the formula [3]:

wherein X and n are the same as defined above, R^(3a) is —C(R^(a))═NRb,in which R^(a) is the same as defined above, and Rb is a hydrogen atomor a protecting group for imidoyl group, or R^(3a) is a protecting groupfor amino group, in the presence of a base, or reacting the compound ofthe formula [2] with a thiolate salt of the compound of the formula [3]to give a compound of the formula [4]:

wherein R^(1a), R², R^(3a), R⁴, X and n are the same as defined above,

followed by reactions which are properly selected

from the removal of the protecting group for hydroxy group for R^(1a),the removal of the protecting group for amino group for R^(3a) and thesubsequent imidoylization reaction of the de-protected amino group, orthe removal of the protecting group for imidoyl group for R^(3a), andthe removal of the protecting group for carboxyl group for R⁴.

The lower alkyl group for R¹, R^(1a), R², R³ or R^(3a) in the aboveformulae [1], [2], [3] and [4] includes alkyl groups having 1 to 5carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl, and n-pentyl.

The lower alkyl group being substituted by a hydroxy group for R¹ orR^(1a) includes alkyl groups having 1 to 5 carbon atoms and beingsubstituted by a hydroxy group, for example, hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxy-l-methylethyl,1-hydroxypropyl, and 2-hydroxypropyl.

The substituent for the substituted lower alkyl group in the presentinvention includes, for example, a hydroxy group; a lower alkoxy group;a lower alkoxy group being substituted by a hydroxy group or an aminogroup; a lower alkylcarbonyloxy group; a lower alkoxycarbonyl group; anamino group; an amino group being substituted by one or two groupselected from a lower alkyl group and a lower alkyl group beingsubstituted by a hydroxy group or an amino group; a guanidino group; aquaternary ammonium group being substituted by three groups selectedfrom a lower alkyl group and a lower alkyl group being substituted by ahydroxy group or an amino group; a carboxyl group; an aminocarbonylgroup; an aminocarbonyl group being substituted by one or two groupsselected from a lower alkyl group and a lower alkyl group beingsubstituted by a hydroxy group or an amino group; a halogen atom; acyano group; an alkylamidino group having 1 to 3 carbon atoms; and aguanidinocarbonyl group. These substituents may optionally be protectedby an appropriate protecting group. The positions of these substituentsmay be any position which is chemically possible, and the substitutioneither at one position or at more positions such as 1 to 3 positions isalso available. The substituted lower alkyl group also includes onesforming a 3- to 7-membered ring via a heteroatom that is a substituent.The 3- to 7-membered ring thus formed includes rings such as aziridine,azetidine, pyrrolidine, piperidine, morpholine, homopiperazine, andtetrahydrofuran.

The protecting group for carboxyl group for R⁴ in the above formulae [2]and [4] may be any conventional protecting groups, but preferably astraight chain or branched chain lower alkyl group having 1 to 5 carbonatoms (e.g., methyl, ethyl, isopropyl, tert-butyl, etc.), ahalogeno-lower alkyl group having 1 to 5 carbon atoms (e.g.,2-iodoethyl, 2,2,2-trichloroethyl), a lower alkoxymethyl group having 1to 5 carbon atoms (e.g., methoxymethyl, ethoxymethyl, isobutoxymethyl),a lower aliphatic acyloxymethyl group having 2 to 5 carbon atoms (e.g.,acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl),a 1-(C₁-C₅)alkoxycarbonyloxyethyl group (e.g.,1-ethoxycarbonyloxyethyl), an aralkyl group (e.g., benzyl,p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl), a lower alkenyl grouphaving 3 to 7 carbon atoms (e.g., allyl, 3-methylallyl), a benzhydrylgroup, or a phthalidyl group.

The protecting group for hydroxy group for R^(1a) in the formulae [2]and [4], and the protecting group for imidoyl group and the protectinggroup for amino group for R^(3a) in the formulae [3] and [4] may be anyconventional ones, but preferably an alkoxycarbonyl group having 1 to 5carbon atoms (e.g., tert-butyloxycarbonyl), a halogenoalkoxycarbonylgroup having 1 to 5 carbon atoms (e.g., 2-iodoethyloxycarbonyl,2,2,2-trichloroethyloxycarbonyl), a substituted or unsubstitutedalkenyloxycarbonyl group having 3 to 7 carbon atoms (e.g.,allyloxycarbonyl), an aralkyloxycarbonyl group (e.g., benzyloxycarbonyl,p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl), or a trialkylsilyl group (e.g.,trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl).

The pharmaceutically acceptable salt of the compound of the aboveformula [1] is a conventional non-toxic salt thereof. Such saltsinclude, as a salt with an intramolecular carboxylic acid, a salt withan inorganic base such as sodium, potassium, calcium, magnesium,ammonium, a salt with an organic base such as triethylammonium,pyridinium, diisopropylammonium, or an intramolecular salt being formedwith a cation at the 3-side chain such as a quaternary ammonium ion. Asa salt with an intramolecular base, a salt with an inorganic acid suchas hydrochloric acid, sulfuric acid, phosphoric acid, or a salt with anorganic acid such as formic acid, acetic acid, oxalic acid,methanesulfonic acid, benzenesulfonic acid can be exemplified.

The non-toxic ester of the compound [1] includes a conventionalpharmaceutically acceptable ester at the 2-carboxyl group of carbapenemantibacterial agents, and may be esters being able to be easilyhydrolyzed in the living body, for example, esters with acetoxymethyl,pivaloyloxymethyl, 1-(ethoxycarbonyloxy)ethyl, and phthalidyl.

The β-lactam compound of the formula [1], a pharmaceutically acceptablesalt thereof, or a non-toxic ester thereof may be in the form of ananhydride thereof, a hydrate thereof, or a solvate thereof.

The process for preparing the present compound is illustrated in moredetail below.

The compound of the formula [4]:

where in R^(1a), R², R^(3a), R⁴, X and n are the same as defined above,can be prepared by reacting a reactive ester of the formula [2]:

wherein R^(1a), R², R⁴ and L are the same as defined above, with amercaptan compound of the formula [3]:

wherein R^(3a), X and n are the same as defined above, in the presenceof a base in an inert solvent, or by reacting a compound of the formula[2] with a thiolate salt of the compound of the formula [3] in an inertsolvent.

The active ester of hydroxy group includes, for example, a substitutedor unsubstituted arylsulfonic acid ester (e.g., benzenesulfonic acidester, p-toluenesulfonic acid ester, p-nitrobenzenesulfonic acid ester,p-bromobenzenesulfonic acid ester, etc.), an alkanesulfonic acid esterhaving 1 to 5 carbon atoms (e.g., methanesulfonic acid ester,ethanesulfonic acid ester, etc.), a halogenoalkanesulfonic acid esterhaving 1 to 5 carbon atoms (e.g., trifluoromethanesulfonic acid ester,etc.), an arylphosphoric acid ester (e.g., diphenylphosphoric acidester, etc.), or a halide compound such as chloride, bromide, iodidewhich is an ester with a hydrogen halide. The preferable reactive esterof hydroxy group may be p-toluenesulfonic acid ester, methanesulfonicacid ester, trifluoromethanesulfonic acid ester, and diphenylphosphoricacid ester.

The inert solvent, which is used in the reaction between the compound[2] and the compound [3] in the presence of a base to give the compound[4], includes, for example, dioxane, tetrahydrofuran, dimethylsulfoxide,dimethylformamide, acetonitrile, benzene, toluene,hexamethylphophoramide, or a mixture of these solvents.

The base includes, for example, an inorganic base such as sodiumcarbonate, potassium carbonate, sodium hydride, potassium hydride, or anorganic base such as pyridine, dimethylaminopyridine, triethylamine,diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).Especially preferable one is DBU. The base should be used in an amountsufficient for carrying out the reaction, and it is usually used in anamount of 1 to 3 equivalents, to the amount of the mercaptan compound[3].

The mercaptan compound [3] should be used in an amount sufficient forcarrying out the reaction, and can be used in a large excess amount, butit is usually used in an amount of 1 to 2 equivalents, to the amount ofthe compound [2].

The reaction is carried out at a temperature from −78° C. to +60° C.,preferably at a temperature from −40° C. to +40° C. Besides, after thereaction is completely over, the product thus obtained is isolated by aconventional organic chemical method.

The inert solvent, which is used in the reaction between the compound[2] and a thiolate salt of the compound [3] to give the compound [4],includes, for example, dioxane, tetrahydrofuran, dimethylsulfoxide,dimethylformamide, acetonitrile, benzene, toluene,hexamethylphosphoramide, or a mixture of these solvents.

The thiolate salt should be used in an amount sufficient for carryingout the reaction, and can be used in a large excess amount, but it isusually used in an amount of 1 to 2 equivalents, to the amount of thecompound [2].

The reaction is carried out at a temperature from −78° C. to +60° C.,more preferably at a temperature from −40° C. to +40° C. After thereaction is completely over, the product thus obtained is isolated by aconventional organic chemical method.

The thiolate salt is prepared from the mercaptan compound [3] and abase. The base includes, for example, an inorganic base (e.g., sodiumhydride, potassium hydride), a metal alkoxide (e.g., potassiumtert-butoxide, sodium methoxide), or a metal amide (e.g., sodium amide,lithium diisopropylamide, lithium disirazide).

The β-lactam compound of the formula [1] is obtained from the compound[4] in a conventional manner, if necessary, by reactions which areproperly selected from the removal of the protecting group for hydroxygroup for R^(1a), the removal of the protecting group for amino groupfor R^(3a) and the subsequent imidoylization reaction of thede-protected amino group, or the removal of the protecting group forimidoyl group for R^(3a), and the removal of the protecting group forcarboxyl group for R⁴.

The removal of a protecting group for hydroxy group for R^(1a), theremoval of a protecting group for imidoyl group for R^(3a), the removalof a protecting group for amino group for R^(3a), and the removal of aprotecting group for carboxyl group for R⁴ are carried out by treatingwith an acid, a base, or a reducing agent, and these methods per se arewell known methods, as disclosed, for example, in T. W. Greene:Protective Groups in Organic Synthesis, J. Wiley & Sons Inc., 1981. Theacid is preferably trifluoroacetic acid, formic acid, boron trifluoride,aluminum chloride, etc., or a mixture of these acids. The base ispreferably an alkali metal carbonate (e.g., sodium carbonate, potassiumcarbonate, etc.), an alkali metal sulfide (e.g., sodium sulfide,potassium sulfide, etc.), or tetrabutylammonium fluoride. The reductionmethod includes, for example, hydrogenation with zinc and acetic acid,hydrogen and palladium-carbon or platinum, etc. The reduction is alsocarried out by using a combination of reducing agents such as palladium(0) or palladium (II) and tri-n-butyltin hydride.

The solvent may be any ones which do not disadvantageously affect thereaction, and includes, for example, water, alcohols (e.g., methanol,ethanol), ethers (e.g., tetrahydrofuran, dioxane), fatty acids (e.g.,acetic acid), or a mixture of these solvents. The reaction can possiblybe suppressed or promoted by properly lowering or raising the reactiontemperature. The preferable reaction temperature is in the range from−30° C. to +40° C. After the reaction is completely over, the productthus obtained can be isolated by a conventional organic chemical method,for example, by neutralizing the reaction mixture, subjecting it tocolumn chromatography on absorption resin, etc., collecting thefractions containing the desired compound, and then followed bylyophilizing the resultant.

The compound of the formula [2] is a well known compound, and isdisclosed, for example, in JP-B-63-55514.

The mercaptan compound [3] can be prepared by conventional variousmethods, for example, by methods disclosed in literatures such as K.Hoffmann, Heterocyclic Chemistry vol. 6 (1953); J. V. Metzger, ibid.,vol. 34 (1979); I. J. Turchi, ibid., vol. 45 (1986); IntersciencePublishers, Inc. or A. R. Katritzky, Advances in Heterocyclic Chemistry,vol. 32 (1982), Academic Press, or by combining these methods. Themercaptan compound [3] is prepared, for example, by the followingreaction scheme.

wherein R⁵ is an imidoyl group protected by a protecting group, or aprotecting group for amino group, and tBu means 1,1-dimethylethyl(tert-butyl) group. Of course, the process for preparing the mercaptancompound [3] should not be construed to be limited to the above ReactionScheme.

The compound of the above-mentioned formula [1] may have optical isomersbased on the asymmetric carbon atoms at the 4-, 5- and 6-positions ofthe carbapenem nucleus, as shown in the following formula:

wherein R¹, R², R³, X and n are the same as defined above, and * meansan asymmetric carbon atom, and these isomers are all convenientlyexpressed by only one formula. However, the present invention should notbe construed to be limited thereto, and includes all isomers and amixture of isomers based on each asymmetric carbon atom. However, thepreferable isomers are ones wherein when R² is a hydrogen atom, the5-carbon atom has an R-configuration such as (5R,6R)-compounds or (5R,6S)-compounds, and when R² is a lower alkyl group, the 4-carbon atom hasan R-configuration and the 5-carbon atom has an S-configuration, such as(4R,5S,6S)-compounds or (4R,5S,6R)-compounds. Moreover, when R¹ is1-hydroxyethyl group, the compound [1] may have isomers having anR-configuration or an S-configuration at the 8-position, as shown in thefollowing formula:

wherein R², R³, X and n are the same as defined above, and thepreferable one is ones having an R-configuration at the 8-position.

Isomers having such configurations are prepared by using eachcorresponding isomer of the starting compound [2].

In addition, the compound [1] is also considered to have tautomers ofthe following formula:

wherein R¹, R² ₁ R^(a), X and n are the same as defined above, but theformula [1] is exemplified as a formula conveniently indicating all ofthese tautomers as well.

The present compounds of the formula [1] are novel β-lactam compoundshaving an azolethio group at the 3-position of the carbapenem nucleus,and a cycloalkenyl group at the 4-position of said azole group, andfurther having an amino group being substituted by various imidoylgroups within said cycloalkenyl ring. These compounds show an excellentantibacterial activity, and are useful as medicaments.

Representative compounds of the formula [1] obtained by the presentinvention are exemplified in the following Table 1.

TABLE 1 [1]

Comp. No. R¹ R² X n R³ 1 CH₃CH(OH)— Me S 2 —C(CH₂CH₃)═NH 2 CH₃CH(OH)— MeS 2 —C(CH₂CH₂CH₃)═NH 3 CH₃CH(OH)— Me S 2 —C(CH₂(CH₃)₂)═NH 4 CH₃CH(OH)—Me S 2 —C(CH₂OH)═NH 5 CH₃CH(OH)— Me S 2 —C(CH₂CH₂OH)═NH 6 CH₃CH(OH)— MeS 2 —C(CH₂NH₂)═NH 7 CH₃CH(OH)— Me S 2 —C(CH₂CONH₂)═NH 8 CH₃CH(OH)— Me S2 —C(CH₂COOCH₃)═NH 9 CH₃CH(OH)— Me S 2 —C(CH₂CH₂Cl)═NH 10 HOCH₂— Me S 2—CH═NH 11 HOCH₂— Me S 2 —C(CH₃)═NH 12 CH₃CH(OH)— H S 2 —CH═NH 13CH₃CH(OH)— H S 2 —C(CH₃)═NH 14 CH₃CH(OH)— Me O 2 —CH═NH 15 CH₃CH(OH)— MeO 2 —C(CH₃)═NH 16 CH₃CH(OH)— Me NH 2 —CH═NH 17 CH₃CH(OH)— Me NH 2—C(CH₃)═NH 18 CH₃CH(OH)— Me S 1 —CH═NH 19 CH₃CH(OH)— Me S 1 —C(CH₃)═NH20 CH₃CH(OH)— Me S 3 —CH═NH 21 CH₃CH(OH)— Me S 3 —C(CH₃)═NH

The compounds as listed in Table 1 have stereoisomers as describedabove, and these exemplified compounds include all of their isomers aswell.

The novel β-lactam compounds represented by the above formula [1] areuseful as medicaments. That is, the compounds [1] exhibit antibacterialactivities against a wide variety of pathogenic bacteria includingGram-positive bacteria such as Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pyogenes, Streptococcus pneumoniae,Enterococcus faecalis, and Gram-negative bacteria such as Escherichiacoli, the genus Proteus, Klebsiella pneumoniae, Haemophilus influenzae,Neisseria gonorrhoeae, the genus Branhamella, and especially exhibitexcellent antibacterial activities against Gram-positive bacteria, aswell as against MRSA and MRCNS.

It is well known that dehydropeptidase-I (DHP-I), a renal enzyme, caneasily hydrolyze carbapenem compounds derived from natural resources,but some of the present compounds [1], which are also carbapenemcompounds, are stable over DHP-I, and can be used alone, but a DHP-Iinhibitor may be used together with the present compound, if necessary.

The present compounds have excellent physiochemical properties, forexample, excellent solubility and stability in water or in an aqueoussolution such as a buffer.

When used as an antibacterial agent in the treatment of infectiousdiseases caused by bacteria, the present compounds are administered, forexample, orally in the form of a tablet, capsule, powder, syrup, etc.,or parenterally such as intravenous injection, intramuscular injection,or intrarectal administration.

The suitable administration forms as mentioned above may be prepared bymixing an active ingredient with a conventional pharmaceuticallyacceptable carrier, excipient, binder, stabilizer, etc. Whenadministered in the form of an injection, a pharmaceutically acceptablebuffering agent, solubilizer, isotonic agent, etc. may be added thereto.

The dosage of the compound [1] varies according to the conditions, ages,weights of the patient, the administration form, the frequency of theadministration, etc., but it is usually in the range of 100 to 3000 mgper day for an adult, which is administered once or divided into severaldosage units. Besides, the dosage of the compound [1] may be increasedor decreased, if necessary.

EXAMPLES

The present invention is illustrated in more detail by Examples, butshould not be construed to be limited thereto.

The following abbreviations are used in Examples.

Ph: Phenyl group

TMS: Trimethylsilyl group

Me: Methyl group

ALOC: 2-Propenyloxycarbonyl group (Allyloxycarbonyl group)

Example 1

a) A solution of(4R,5R,6S,8R)-(2-propenyl)-3-(diphenylphosphoryloxy)-4-methyl-6-(1-(trimethylsilyloxy)-ethyl)-1-azabicyclo[3.2.0]hept-2-en-7-one-2-carboxylate(2.02 g, 3.53 mmol) in acetonitrile (5 ml) is stirred at 0° C., andthereto is added gradually a suspension of a thiolate salt, which isprepared by adding2-mercapto-4-(4-(1-(2-propenyl)oxycarbonyl-1,2,5,6-tetrahydropyridyl))-thiazole(997 mg, 3.53 mmol) into a suspension of sodium hydride (101 mg, 4.23mmol) in THF (30 ml), and the mixture is allowed to stand at 5° C. for72 hours. The mixture is stirred at 0° C., and the pH value of themixture is adjusted to pH 3 with iN hydrochloric acid, and the reactionmixture is stirred for 30 minutes.

The pH value of the reaction mixture is adjusted to pH 8.5 with asaturated aqueous sodium hydrogen carbonate solution, and the mixture isdiluted with ethyl acetate. The organic layer is dried over magnesiumsulfate, concentrated under reduced pressure, and the residue thusobtained is purified by silica gel column chromatography (solvent;hexane:ethyl acetate=10:2) to give(4R,5S,6S,8R)-(2-propenyl)-3-(4-(4-(1-(2-propenyl)oxy-carbonyl-1,2,5,6-tetrahydropyridyl))thiazol-2-ylthio)-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3.2.0]hepto-2-en-7-one-2-carboxylate(810 mg, yield; 41%), as a pale yellow amorphous.

¹H-NMR δ (CDCl₃):1.09 (3H, d, J=6.6Hz), 1.23 (3H, d, J=7.3Hz), 2.51 (2H,brs), 3.26 (1H, m), 3.50 (1H, m), 3.69 (2H, m), 4.10 (2H, m), 4.20 (2H,m), 4.6-4.8 (4H, m), 5.0-5.5 (4H, m), 5.8-6.0 (2H, m), 6.66 (1H, s),7.36 (1H, s)

b) (4R,5S,6S,8R)-(2-Propenyl)-3-(4-(4-(1-(2-propenyl) oxycarbonyl-1,2,5, 6-tetrahydropyridyl) )thiazol-2-ylthio)-4-methyl-6- (1-hydroxyethyl)-1-azabicyclo [3.2.0]-hepto-2-en-7-one-2-carboxylate (610 mg, 1.09 mmol)is dissolved in m-chlorobenzene (15 g), and thereto is added aniline(2.03 g, 2.2 mmol). The mixture is stirred at room temperature undernitrogen atmosphere for 30 minutes. The reaction mixture is cooled to 0°C., and thereto is added tetrakistriphenylphosphinepalladium (62.9 mg,0.054 mmol), and the mixture is further stirred for one hour.

To the reaction mixture are added a saturated aqueous sodium chloridesolution (10 ml) and an aqueous sodium hydrogen carbonate solution (pH8.0-8.5, 10 ml), and the mixture is washed twice with dichloromethane.The aqueous layer is concentrated under reduced pressure to remove thesolvent, and the resultant is purified by polymer chromatography(CHP-20P). The fractions eluted with 4-8% aqueous THF solution arecombined and lyophilized to give (4R,5S,6S, 8R)-(3-(4-(4-(1,2,5,6-tetrahydropyridinio) )thiazol-2-yl-thio)-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3.2.0]hepto-2-en-7-one-2-carboxylate (80mg, yield 18%), as a white amorphous.

UV max nm (H₂O):313, 247 (sh)

IR max cm⁻¹ (KBr):3388, 1762, 1599, 1388

¹H-NMR δ (D₂O):1.10 (3H, d, J=7.3Hz), 1.27 (3H, d, J=6.6Hz), 2.83 (2H,m), 3.32 (1H, m), 3.51 (3H, m), 3.93 (2H, m), 4.26 (2H, m), 6.53 (1H,brs), 7.61 (1H, s)

c)(4R,5S,6S,8R)-(3-(4-(4-(1,2,5,6-Tetrahydro-pyridinio))thiazol-2-ylthio)-4-methyl-6-(1-hydroxyethyl)-1-5azabicyclo[3.2.0]hepto-2-en-7-one-2-carboxylate (145 mg, 0.36 mmol) isdissolved in 0.1M sodium phosphate buffer (pH 7.0, 4 ml), and thereto isadded at 0° C. 1N aqueous sodium hydroxide solution to adjust the pHvalue thereof to pH 8.5. To the mixture is added benzyl formimidatehydrochloride (389 mg, 2.88 mmol), and the pH value thereof is adjustedto pH 8.5 with iN aqueous sodium hydroxide solution, and stirred for twohours. The pH value of the reaction solution is adjusted to pH 7.0 withiN hydrochloric acid, and the mixture is washed twice withdichloromethane. The aqueous layer is concentrated under reducedpressure to remove the solvent, and the resultant is purified by polymerchromatography (CHP-20P). The fractions eluted with 4% aqueous THFsolution are combined, and lyophilized to give(4R,5S,6S,8R)-3-(4-(4-(1-formimidoyl-1,2,5,6-tetrahydropyridinio))thiazol-2-ylthio)-4-methyl-6-(1-hydroxyethyl)-1-azabicyclo[3.2.0]hepto-2-en-7-one-2-carboxylate(40 mg, yield; 25%), as a white amorphous.

UV max nm (H₂O):314, 227

IR max cm⁻¹ (KBr):3401, 1764, 1710, 1596, 1384

¹H-NMR δ (D₂O):0.98 (3H, d, J=7.3Hz), 1.19 (3H, d, J=6.3Hz), 2.64 (2H,brs), 3.18-3.24 (1H, m), 3.38-3.41 (1H, m), 3.73-3.83 (2H, m), 4.13-4.19(4H, m), 6.40 and 6.45 (totally 1H, each s), 7.45 and 7.46 (totally 1H,each s), 7.90 and 7.91 (totally 1H, each s).

Example 2

(4R,5S,6S,8R)-3-(4-(4-(l-Acetoimidoyl-1,2,5,6-tetra-hydropyridinio))thiazol-2-ylthio)-4-methyl-6-(1-hydroxy-ethyl)-1-azabicyclo[3.2.0]hepto-2-en-7-one-2-carboxylateis obtained as a white amorphous in the same manner as in Example 1except that ethyl acetoimidate hydrochloride is used in Step (c).

UV max nm (H₂O):313, 229;

IR max cm⁻ (KBr):3354, 1762, 1603, 1381;

¹H-NMR δ (D₂O):1.05 (3H, d, J=7.3Hz), 1.25 (3H, d, J=6.3Hz), 2.37 and2.41 (totally 3H, each s), 2.69 (2H, brs), 3.29 (1H, m), 3.47 (1H, m),3.78-3.83 (2H, m), 4.20-4.34 (4H, m), 6.47 (1H, s), 7.53 and 7.54(totally 1H, each s).

Reference Example

One example of a process for preparing the mercapto compound of theformula [3] is explained below. The following abbreviations are used inthe following Reference Example.

Bn: Benzyl group

Et: Ethyl group

ALOC: 2-Propenyloxycarbonyl group (allyloxycarbonyl group)

a) 1-Benzyl-4-ethoxycarbonylpyridinium bromide (580 g, 1.80 mol) issuspended in a mixture of ethanol (900 ml) and water (900 ml), andthereto is added sodium borohydride (74.9 g, 1.98 mol) in portions whilethe temperature of the reaction mixture is kept at a temperature below10° C. The reaction mixture is stirred at 10° C. for three hours. Thereaction mixture is concentrated under reduced pressure, and the residueis dissolved in chloroform. The mixture is washed with water, dried oversodium sulfate, and concentrated under reduced pressure to remove thesolvent. The residue is dissolved in chloroform (500 ml), and thereto isadded dropwise ethyl chloroformate (215 g, 1.98 mol) under reflux over aperiod of one hour. The mixture is further refluxed for 30 minutes, andconcentrated under reduced pressure. To the residue are added conc.hydrochloric acid (504 g) and water (150 g), and the mixture is refluxedfor 10 hours. The reaction mixture is cooled to room temperature, andconcentrated under reduced pressure. To the residue is added isopropylalcohol (500 ml), and the mixture is stirred. The precipitated crystalsare collected by filtration with suction, and washed with a small amountof ether, and dried with air to give 1,2,5,6-tetrahydroisonicotinic acidhydrochloride (151.7 g, 52%), as white crystals.

¹H-NMR δ (D₂0):2.48 (2H, m), 3.29 (2H, m), 3.79 (2H, m), 6.81 (1H, m)

b) 1,2,5,6-Tetrahydroisonicotinic acid hydrochloride (150 g, 0.92 mol)is added to an aqueous solution of sodium carbonate (194 g, 1.83 mol) inwater (920 ml), and thereto is added dropwise 2-propenyl chloroformate(116 g, 0.96 mol) under ice-cooling over a period of one hour. After theaddition, the mixture is further stirred under icecooling for fivehours. Separately, conc. hydrochloric acid (191 g) and dichloromethane(1000 ml) are cooled, and thereto is added dropwise the above reactionmixture in portions. The mixture is separated, and the aqueous layer isextracted twice with dichloromethane. The organic layers are combined,washed with a saturated aqueous sodium chloride solution, dried overmagnesium sulfate, and concentrated under reduced pressure to remove thesolvent to give1-(2-propenyloxycarbonyl)-1,2,5,6-tetrahydro-isonicotinic acid (198.9 g,quant.) as a white solid.

¹H-NMR δ (CDCl₃):2.42 (2H, m), 3.60 (2H, m), 4.17 (2H, m), 4.63 (2H, m),5.19-5.34 (2H, m), 5.94 (1H, m), 7.01 (1H, s), 10.47 (1H, brs)

c) 1-(2-Propenyloxycarbonyl)-1,2,5,6-tetrahydro-isonicotinic acid (114.2g, 0.54 mol) is dissolved in THF (450 ml), and thereto is addedcarbonydiimidazole (105 g, 0.65 mol) at room temperature, and themixture is stirred for 30 minutes. The mixture is added dropwise to asolution of magnesium di(mono-tert-butylmalonate) (129.6 g, 0.38 mol) inTHF (800 ml) at room temperature, and then the mixture is allowed tostand overnight. The reaction mixture is diluted with ethyl acetate, andwashed successively with 1N hydrochloric acid, a saturated aqueoussodium hydrogen carbonate solution, and a saturated aqueous sodiumchloride solution, dried over magnesium sulfate, concentrated to removethe solvent, to givetert-butyl-4-(1-(2-propenyl)oxycarbonyl-1,2,5,6-tetrahydropyridyl)-carbonylacetate(141.4 g, 86%) as pale yellow oil.

¹H-NMR δ (CDCl₃):1.45-1.65 (9H, m), 2.41 (2H, m), 3.56-3.70 (4H, m),4.21 (2H, m), 4.63 (2H, m), 5.20-5.35 (2H, m), 5.94 (1H, m), 6.77 (1H,s)

d)tert-Butyl-4-(1-(2-propenyl)oxycarbonyl-1,2,5,6-tetrahydropyridyl)carbonylacetate(141.1 g, 0.46 mol) is dissolved in dichloromethane (400 ml), andthereto is added tert-butanol (33.8 g, 0.46 mol). The mixture is cooledto −15 to −20° C., and thereto is added gradually dropwise sulfurylchloride (38.8 ml, 0.48 mol) with stirring. Then, the mixture is stirredfor one hour. The reaction mixture is warmed to room temperature, andthereto is added a saturated aqueous sodium hydrogen carbonate solution.The mixture is separated, and the organic layer is dried over magnesiumsulfate, and concentrated to remove the solvent. The residue isdissolved in ethylene dichloride (350 ml), and thereto is addedmethanesulfonic acid (12.4 g, 0.13 mol). The mixture is warmed to 70°C., and stirred for four hours. The reaction mixture is cooled to roomtemperature, and thereto is added a saturated aqueous sodium hydrogencarbonate solution. The mixture is extracted with ethyl acetate, and theorganic layer is dried over magnesium sulfate, and concentrated toremove the solvent. The residue is purified by silica gel columnchromatography (hexane:ethyl acetate=10:3) to givechloromethyl-4-(1-(2-propenyl)oxycarbonyl-1,2,5,6-tetrahydropyridyl)ketone(52.9 g, 47%) as a pale yellow solid.

¹H-NMR δ (CDCl₃):2.44 (2H, m), 3.60 (2H, m), 4.27 (2H, m), 4.43 (2H, s),4.63 (2H, m), 5.22-5.38 (2H, m), 5.95 (1H, m), 6.85 (1H, s)

e)Chloromethyl-4-(1-(2-propenyl)oxycarbonyl-1,2,5,6-tetrahydropyridyl)ketone(29.4 g, 0.12 mol) is dissolved in a mixture of ethanol (200 ml) and THF(200 ml), and thereto is added ammonium dithiocarbamate (14.2 g, 0.14mol). The mixture is stirred at room temperature for two hours. Thereaction mixture is concentrated under reduced pressure, and to theresidue is added dichloromethane. The mixture is washed with water and asaturated aqueous sodium chloride solution, dried over magnesiumsulfate, and concentrated to remove the solvent. The residue isdissolved in a mixture of ethanol (200 ml) and THF (200 ml), and themixture is refluxed with stirring for two hours. The reaction mixture iscooled to room temperature, and concentrated under reduced pressure atroom temperature to give2-mercapto-4-(4-(1-(2-propenyl)oxycarbonyl-1,2,5,6-tetrahydropyridyl))thiazole(34 g, quant.).

¹H-NMR δ (CDCl₃):2.41 (2H, m), 3.68 (2H, m), 4.19 (2H, s), 4.64 (2H, m),5.21-5.35 (2H, m), 5.95 (1H, m), 6.28 (1H, s), 6.44 (1H, s), 11.98 (1H,brs)

What is claimed is:
 1. A β-lactam compound of the formula (1):

wherein R¹ is a lower alkyl group or a lower alkyl group beingsubstituted by a hydroxy group, R² is a hydrogen atom or a lower alkylgroup, X is 0, S or NH, n is 1 to 3, and R³ is —C(R^(a))═NH, in whichR^(a) is a hydrogen atom, a lower alkyl group, or a substituted loweralkyl group, or a pharmaceutically acceptable salt thereof, or anon-toxic ester thereof, wherein the non-toxic ester is apharmaceutically acceptable ester at the 2-carboxyl group, selected fromthe group consisting of esters with acetoxymethyl, pivaloyloxymethyl,1-(ethoxycarbonyloxy)ethyl and phthalidyl.
 2. The β-lactam compoundaccording to claim 1, wherein n is 2 or a pharmaceutically acceptablesalt thereof, or a non-toxic ester thereof, wherein the non-toxic esteris a pharmaceutically acceptable ester at the 2-carboxyl group, selectedfrom the group consisting of esters with acetoxymethyl,pivaloyloxymethyl, 1-(ethoxycarbonyloxy)ethyl and phthalidyl.
 3. Theβ-lactam compound according to claim 2, wherein X is a sulfur atom or anoxygen atom, or a pharmaceutically acceptable salt thereof, or anon-toxic ester thereof, wherein the non-toxic ester is apharmaceutically acceptable ester at the 2-carboxyl group, selected fromthe group consisting of esters with acetoxymethyl, pivaloyloxymethyl,1-(ethoxycarbonyloxy)ethyl and phthalidyl.
 4. The β-lactam compoundaccording to claim 3, wherein R¹ is 1-(R)-hydroxyethyl, or apharmaceutically acceptable salt thereof, or a non-toxic ester thereof,wherein the non-toxic ester is a pharmaceutically acceptable ester atthe 2-carboxyl group, selected from the group consisting of esters withacetoxymethyl, pivaloyloxymethyl, 1-(ethoxycarbonyloxy)ethyl andphthalidyl.
 5. The β-lactam compound according to claim 4, wherein X isa sulfur atom, or a pharmaceutically acceptable salt thereof, or anon-toxic ester thereof, wherein the non-toxic ester is apharmaceutically acceptable ester at the 2-carboxyl group, selected fromthe group consisting of esters with acetoxymethyl, pivaloyloxymethyl,1-(ethoxycarbonyloxy)ethyl and phthalidyl.
 6. A pharmaceuticalcomposition useful as an antibacterial agent, which comprises as anactive ingredient the β-lactam compound of the formula (1) according toclaim 1, 2, 3, 4, or 5, or the pharmaceutically acceptable salt thereof,or the non-toxic ester thereof, in admixture with a pharmaceuticallyacceptable carrier or dilutent, wherein the non-toxic ester is apharmaceutically acceptable ester at the 2-carboxyl group, selected fromthe group consisting of esters with acetoxymethyl, pivaloyloxymethyl,1-(ethoxycarbonyloxy)ethyl and phthalidyl.
 7. A process for preparing aβ-lactam compound of the formula (1):

wherein R¹ is a lower alkyl group or a lower alkyl group beingsubstituted by a hydroxy group, R² is a hydrogen atom or a lower alkylgroup, X is 0, S or NH, n is 1 to 3, and R³ is —C(R^(a))═NH, in whichR^(a) is a hydrogen atom, a lower alkyl group, or a substituted loweralkyl group, or a pharmaceutically acceptable salt thereof, or anon-toxic ester thereof, wherein the non-toxic ester is apharmaceutically acceptable ester at the 2-carboxyl group, selected fromthe group consisting of esters with acetoxymethyl, pivaloyloxymethyl,1-(ethoxycarbonyloxy)ethyl and phthalidyl, which comprises reacting acompound of the formula (2):

wherein R² is the same as defined above, R^(1a) is a lower alkyl group,a lower alkyl group being substituted by a hydroxy group, or a loweralkyl group being substituted by a hydroxy group protected by aprotecting group, R⁴ is a protecting group for carboxyl group, and L isan active ester of hydroxy group, with a compound of the formula (3):

wherein X and n are the same as defined above, R^(3a) is —C(R^(a))═NRb,in which R^(a) is the same as defined above, and Rb is a hydrogen atomor a protecting group for imidoyl group, or R^(3a) is a protecting groupfor amino group, in the presence of a base, or reacting the compound ofthe formula (2) with a thiolate salt of the compound of the formula (3)to give a compound of the formula (4):

wherein R^(1a), R², R^(3a), R⁴, X and n are the same as defined above,followed by reactions which are selected from the removal of theprotecting group for hydroxy group for R^(1a), the removal of theprotecting group for amino group for R^(3a) and the subsequentimidoylization reaction of the de-protected amino group, or the removalof the protecting group for imidoyl group for R^(3a), and the removal ofthe protection group R⁴ for carboxyl group.